Lethal Exposure Research Articles

INTRATRACHEAL SALINE BOLUS INCREASES SURVIVABILITY IN RABBITS EXPOSED TO LETHAL DOSE OF INHALED HYDROGEN SULFIDE

SESSION TITLE: Pharmacotherapeutics Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: Hydrogen Sulfide (H2S) is a highly toxic, flammable, and colorless gas. In addition to industrial accident risks, H2S has been identified as a potential terrorist chemical and military threat agent for use in mass-casualty events, with no effective antidotes. In this study, we investigate the effects of a small volume of intratracheal (IT) slow-drip saline versus IT fast bolus saline treatment to prolong survival in H2S exposure in an animal model for potential treatments during mass-casualty crises. METHODS: The effects of IT saline in H2S exposure were tested on 49 New Zealand white male rabbits. Animals were anesthetized and intubated, but not ventilated. H2S gas (2000 ppm) was delivered along with a 1.5% isoflurane in air mixture (2.25 liters/min)for 30 minutes.Three groups were compared: no saline into endotracheal tube control(n=16), IT slow saline drip (n=17) and fast IT saline bolus group (n=16). Animals in IT slow drip saline group received 3cc of saline in 3 minutes, while fast saline bolus group animals received it in less than 20 seconds. Both treatment groups were compared to the control group. Systemic blood pressure and heart rate were continuously monitored.The changes in tissue oxy and deoxy hemoglobin concentrations were measured non-invasively over the brain region using continuous wave near infrared spectroscopy. Animals were deemed survivors at 60 minutes from the start of toxic exposure. Animals were sacrificed when systolic blood pressure dropped below 20 mmHg. RESULTS: The average weight for all animals(n=49) in this study was 3.88 ± 0.35 kg, mean ± SD. The survival rate of the fast saline, slow saline, and the control groups were 56.25%, and,23.53%, and 25%.Log-rank (Mantel-Cox) test showed that the survivability in the treatment groups and control groups are significantly different (p=0.0417). Slow saline group versus fast saline group difference was also significant (p=0.0140). The slow saline group compared to control group results were not different (p=0.7031). CONCLUSIONS: The rapid administration of a small volume of saline bolus given via IT route was effective in prolonging survival in lethal H2S exposure when given rapidly. These findings appear likely due to the rapid bolus stimulating cough and breathing responses in apneic animals, in contrast to the slow dripping of a similar amount of saline.While the stimulation of breathing leads to inhalation of more H2S in this model, improvement in hypoxemia in these animals appears to be of greater importance than the risk of additional H2S exposure.Thus, we propose that the hypoxemia from H2S-induced respiratory depression is a major contributor to mortality compared to the metabolic inhibition of cytochromes from H2S in this scenario. CLINICAL IMPLICATIONS: This has potential implications for the treatment victims of H2S exposure who cannot be removed from the exposure site as this commonly occurs in mass-casualty events. DISCLOSURES: No relevant relationships by vikhyat bebarta, source=Web Response No relevant relationships by Gerry Boss, source=Web Response No relevant relationships by Matthew Brenner, source=Web Response No relevant relationships by Tanya Burney, source=Web Response No relevant relationships by Adriano Chan, source=Web Response No relevant relationships by Melina Doosty, source=Web Response No relevant relationships by Philippe Haouzi, source=Web Response No relevant relationships by Tara Hendry-Hofer, source=Web Response No relevant relationships by Jangwoen Lee, source=Web Response No relevant relationships by Sari Mahon, source=Web Response No relevant relationships by David Mukai, source=Web Response No relevant relationships by George Philipopoulos, source=Web Response

Survival recovery rates by six clonal lineages of Daphnia longispina after intermittent exposures to copper

Natural populations are commonly exposed to sequential pulses of contaminants. Accordingly, this study aimed at testing the existence of an association between the tolerance to lethal levels of copper (Cu) and the survival recovery ability from pulsed partially lethal copper exposures in six clonal lineages of Daphnia longispina. It was hypothesized that the most tolerant genotypes would be the ones exhibiting a faster survival recovery from a pulsed contaminant exposure. For each clonal lineage, the intensity of pulses corresponded to the respective concentration of Cu causing 30% of mortality after 24h of exposure (LC30,24h). The initial hypothesis was not corroborated: obtained results showed no association between survival recovery and lethal tolerance to Cu. Nevertheless, some patterns could be detected. Firstly, the most sensitive lineages to lethal levels of copper revealed a faster survival recovery from a first Cu pulse comparatively to the most tolerant ones, though they were the most sensitive to a second pulse exposure. Secondly, the most tolerant lineages, though being more tolerant to a second exposure, exhibited the lowest survival recovery capacity after exposure to a first pulse of Cu. However, differences in the survival recovery capacity of the six clonal lineages after the exposure to the two pulses of Cu were not observed. Increasing the duration of the recovery period from 24h to 72h did not significantly alter mortality rates, except for the most sensitive and most tolerant clonal lineages. The results here obtained suggests that standard lethality assays may sub-estimate the toxicity of chemicals under realistic exposure scenarios, since sequential pulses are not infrequent in natural conditions.

Forcing Seasonality of Influenza-like Epidemics with Daily Solar Resonance.

SummarySeasonality of acute viral respiratory diseases is a well-known and yet not fully understood phenomenon. Several models have been proposed to explain the regularity of yearly recurring outbreaks and the phase differences observed at different latitudes on the Earth. Such models consider known internal causes, primarily the periodic emergence of new virus variants that evade the host immune response. Yet, this alone is generally unable to explain the regularity of recurrences and the observed phase differences.Here we show that seasonality of viral respiratory diseases, as well as its distribution with latitude on the Earth, can be fully explained by the virucidal properties of UV-B and UV-A solar photons through a daily, minute-scale, resonant forcing mechanism. Such an induced periodicity can last, virtually unperturbed, from tens to hundreds of cycles, and even in the presence of internal dynamics (host's loss of immunity) much slower than seasonal will, on a long period, generate seasonal oscillations.

Redefining the Clostridioides difficile σB Regulon: σB Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide.

In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile, σB has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allows for the refinement of the previously proposed σB regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σB was demonstrated through in vitro runoff transcription of specific target genes (cd0350, cd3614, cd3605, and cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to σB-dependent gene activation.IMPORTANCE Sigma B is the alternative sigma factor governing stress response in many Gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together, our data suggest that σB is a key player in dealing with toxic radicals.

Optical tracking and laser-induced mortality of insects during flight

Addressing the need for novel insect observation and control tools, the Photonic Fence detects and tracks mosquitoes and other flying insects and can apply lethal doses of laser light to them. Previously, we determined lethal exposure levels for a variety of lasers and pulse conditions on anesthetized Anopheles stephensi mosquitoes. In this work, similar studies were performed while the subjects were freely flying within transparent cages two meters from the optical system; a proof-of-principle demonstration of a 30 m system was also performed. From the dose–response curves of mortality data created as a function of various beam diameter, pulse width, and power conditions at visible and near-infrared wavelengths, the visible wavelengths required significantly lower laser exposure than near infrared wavelengths to disable subjects, though near infrared sources remain attractive given their cost and retina safety. The flight behavior of the subjects and the performance of the tracking system were found to have no impact on the mortality outcomes for pulse durations up to 25 ms, which appears to be the ideal duration to minimize required laser power. The results of this study affirm the practicality of using optical approaches to protect people and crops from pestilent flying insects.

Measured and predicted acute toxicity of phenanthrene and MC252 crude oil to vertically migrating deep-sea crustaceans.

Deep-water column micronekton play a key role in oceanic food webs and represent an important trophic link between deep- and shallow-water ecosystems. Thus, the potential impacts of sub-surface hydrocarbon plumes on these organisms are critical to developing a more complete understanding of ocean-wide effects resulting from deep-sea oil spills. This work was designed to advance the understanding of hydrocarbon toxicity in several ecologically important deep-sea micronekton species using controlled laboratory exposures aimed at determining lethal threshold exposure levels. The current study confirmed the results previously determined for five deep-sea micronekton by measuring lethal threshold levels for phenanthrene between 81.2 and 277.5 μg/L. These results were used to calibrate the target lipid model and to calculate a critical target lipid body burden for each species. In addition, an oil solubility model was used to predict the acute toxicity of MC252 crude oil to vertically migrating crustaceans, Janicella spinacauda and Euphausiidae spp., and to compare the predictions with results of a 48-h constant exposure toxicity test with passive-dosing. Results confirmed that the tested deep-sea micronekton appear more sensitive than many other organisms when exposed to dissolved oil, but baseline stress complicated interpretation of results.

Dichotomous engagement of HDAC3 activity governs inflammatory responses.

Histone deacetylase 3 (HDAC3) is unique among the HDAC superfamily of chromatin modifiers that silence transcription through enzymatic modification of histones because interaction with nuclear receptor corepressors (NCoR1/2) is required for engagement of its catalytic activity1–3. However, loss of HDAC3 also represses transcription4–8. Here we report that, during lipopolysaccharide (LPS) activation of macrophages, recruitment of HDAC3 to ATF2-bound sites without NCoR1/2 non-canonically activates inflammatory gene expression. By contrast, HDAC3 deacetylase activity is selectively engaged at ATF3-bound sites that suppress toll-like receptor (TLR) signaling. Deletion of HDAC3 in macrophages safeguards mice from lethal exposure to LPS, but this protection is not conferred by genetic or pharmacological abolition of HDAC3 catalytic activity. Thus, HDAC3 is a dichotomous transcriptional activator and repressor whose non-canonical deacetylase-independent functions are vital for the innate immune system.

A SNP of the hemocyanin gene (LvHc) is a marker for high growth and ammonia-tolerance in Pacific white shrimp Litopenaeus vannamei

Expression levels of hemocyanin (LvHc), activating transcription factor 4 (LvAtf4), glutathione S-transferase (LvGst), caspase 2 (LvCasp2) and anti-lipopolysaccharide factor (LvAlf) were examined in the hepatopancreas of Pacific white shrimp Litopenaeus vannamei juveniles exposed to a lethal concentration of ammonia-N (32.15 mg/l). The expression levels of all transcripts except LvAlf were significantly greater (P < 0.05) in tolerant shrimp (Lv-AT; N = 30) that survived up to 72 h post treatment (hpt) than in susceptible shrimp (Lv-AS24 and Lv-AS72; N = 45 and 15), that died within 24 h or between 24 and 72 hpt, respectively. Subsequently, effects of non-lethal concentrations of ammonia-N (control, 10 and 20 mg/l) on the expression of LvHc in juvenile shrimp were examined. Compared to the control, expression levels of LvHc transcripts in hemocytes and the hepatopancreas of tested shrimp changed after exposure to ammonia-N. One SNP (C > T545) was found in the LvHc322 gene segment. Real-time PCR amplification of specific alleles (real-time PASA) was developed for detection of C > T545 genotypes. Juveniles in the lethal exposure test that carried a C/T545 genotype showed a greater average body weight and total length (8.46 ± 0.36 g and 10.05 ± 0.16 cm) than those with a C/C545 genotype (7.48 ± 0.31 g and 9.60 ± 0.13 cm) (P < 0.05). Similar results were found in the second generation (G2) of a growth-improved stock (3 and 4 families of BIOTEC-G2-L1 and BIOTEC-G2-L2) and in commercially farmed shrimp (2 groups). Accordingly, expression levels and SNP of LvHc can serve as markers for selection high growth performance in ammonia-tolerant L. vannamei.

TIGAR/AP-1 axis accelerates the division of Lgr5\u2212 reserve intestinal stem cells to reestablish intestinal architecture after lethal radiation

During radiologic or nuclear accidents, high-dose ionizing radiation (IR) can cause gastrointestinal syndrome (GIS), a deadly disorder that urgently needs effective therapy. Unfortunately, current treatments based on natural products and antioxidants have shown very limited effects in alleviating deadly GIS. Reserve intestinal stem cells (ISCs) and secretory progenitor cells are both reported to replenish damaged cells and contribute to crypt regeneration. However, the suppressed β-catenin/c-MYC axis within these slow-cycling cells leads to limited regenerative response to restore intestinal integrity during fatal accidental injury. Current study demonstrates that post-IR overexpression of TIGAR, a critical downstream target of c-MYC in mouse intestine, mounts a hyperplastic response in Bmi1-creERT+ reserve ISCs, and thus rescues mice from lethal IR exposure. Critically, by eliminating damaging reactive oxygen species (ROS) yet retaining the proliferative ROS signals, TIGAR-overexpression enhances the activity of activator protein 1, which is indispensable for initiating reserve-ISC division after lethal radiation. In addition, it is identified that TIGAR-induction exclusively gears the Lgr5− subpopulation of reserve ISCs to regenerate crypts, and intestinal TIGAR-overexpression displays equivalent intestinal reconstruction to reserve-ISC-restricted TIGAR-induction. Our findings imply that precise administrations toward Lgr5− reserve ISCs are promising strategies for unpredictable lethal injury, and TIGAR can be employed as a therapeutic target for unexpected radiation-induced GIS.

Sublethal exposure to deltamethrin impairs maternal egg care in the European earwig Forficula auricularia

The application of pesticides typically leads to lethal and sublethal exposure of non-target insects. Whereas our current understanding of these sublethal effects typically focuses on reproductive and physiological parameters, recent works emphasize that sublethal effects on behaviors such as maternal care could be of major importance in non-target species. However, it remained unknown whether these sublethal effects occur in insects. Here, we tested if exposure to sublethal doses of deltamethrin - a pyrethroid insecticide commonly used in crops - alters the expression of maternal egg care in females of the European earwig Forficula auricularia, a predator insect and pest control. Our results first reveal that deltamethrin exposure impaired the expression of three forms of maternal egg care: It decreased the likelihood of mothers to gather their otherwise scattered clutch of eggs, increased the time during which the female abandoned the clutch after a predator attack and reduced egg grooming duration. These sublethal effects did not reflect a lower activity of deltamethrin-exposed females, as these females increased their expression of self-grooming, and deltamethrin exposure did not affect females’ exploration and mobility. Finally, we found that the negative effects of deltamethrin on egg care did not modify egg development, hatching rate and juvenile weight, possibly due to the transient effects of deltamethrin on maternal behaviors. Overall, our results reveal that sublethal exposure to a pesticide may diminish maternal egg care in a natural pest control and call for the integration of this measurement in assays on pesticides application.

Lethal and sublethal exposure of Hemichromis bimaculatus (Gill, 1862) to malachite green and possible implications for ornamental fish.

Malachite green (MG) is a triphenylmethane dye that is widely used in aquaculture as a fungicide, bactericide, ectoparasiticide, and antiprotozoal. There is great debate regarding the potential for this compound to trigger adverse effects. Here, we review the previous findings and then evaluate the lethal and sublethal effects of MG in the species Hemichromis bimaculatus (jewelfish). The lethal concentration for 50% of the fish in 96h was 1mg/L. We observed a dose-dependent increase in the percentage of fish mortality as well as physical and behavioral changes. We further found that the highest tested sublethal dose significantly increased the DNA damage index identified using the comet assay (74.97 ± 13.8 at a significant level of P< 0.05 for the 0.75mg/L concentration), but did not significantly alter the results of the micronucleus test. Although our results suggest that MG confers risks on exposed fish, the findings were significant only at the highest exposure concentration (0.75mg/L). At lower concentrations (0.25mg/L and 0.5mg/L), no adverse effect was observed. The maximum MG concentration recommended for use in ornamental fish farming is 0.2mg/L. Therefore, our results suggest that, specifically for the parameters analyzed in this work, MG does not have any adverse effect when users strictly adhere to the recommended concentration criteria for ornamental fish.

Mosquito larvae that survive a heat spike are less sensitive to subsequent exposure to the pesticide chlorpyrifos

While extreme high temperatures are an important aspect of global warming, their effects on organisms are relatively understudied, especially in ecotoxicology. Sequential exposure to heat spikes and pesticides is a realistic scenario as both are typically transient stressors and are expected to further increase in frequency under global warming. We tested the effects of exposure to a lethal heat spike and subsequently to an ecologically relevant lethal pulse exposure of the pesticide chlorpyrifos in the larvae of mosquito Culex pipiens. The heat spike caused direct and delayed mortality, and resulted in a higher heat tolerance and activity of acetylcholinesterase, and a lower fat content in the survivors. The chlorpyrifos exposure caused mortality, accelerated growth rate, and decreased the heat tolerance and the activity of acetylcholinesterase. The preceding heat spike did not change how chlorpyrifos reduced the heat tolerance. Notably, the preceding heat spike did lower the lethal effect of the pesticide, which makes an important novel finding at the interface of ecotoxicology and global change biology, and adds a new dimension to the “climate-induced toxicant sensitivity” (CITS) concept. This may be due to both survival selection and cross-tolerance, and therefore likely a widespread phenomenon. Our results emphasize the importance of including extreme high temperatures as an important transient global change stressor in ecotoxicology.

Consequences of a short term, sub lethal pesticide exposure early in life on survival and immunity in the honeybee ( Apis mellifera )

Consequences of a short term, sub lethal pesticide exposure early in life on survival and immunity in the honeybee ( Apis mellifera )

Delayed effects of acute whole body lethal radiation exposure in mice pre-treated with BBT-059

The threat of nuclear exposure is heightened and it is imperative to identify potential countermeasures for acute radiation syndrome. Currently no countermeasures have been approved for prophylactic administration. Effective countermeasures should function to increase survival in the short term as well as to increase the overall prognosis of an exposed individual long term. Here we describe the use of a promising radiation countermeasure, BBT-059, and the results of a long term mouse study (up to 12 months) in the male CD2F1 strain using 60Co gamma irradiation (~0.6 Gy/min, 7.5–12.5 Gy). We report the dose reduction factor of 1.28 for BBT-059 (0.3 mg/kg) compared to control administered 24 h prior to irradiation. In the long term study animals showed accelerated recovery in peripheral blood cell counts, bone marrow colony forming units, sternal cellularity and megakaryocyte numbers in drug treated mice compared to formulation buffer. In addition, increased senescence was observed in the kidneys of animals administered control or drug and exposed to the highest doses of radiation. Decreased levels of E-cadherin, LaminB1 and increased levels of Cyc-D and p21 in spleen lysates were observed in animals administered control. Taken together the results indicate a high level of protection following BBT-059 administration in mice exposed to lethal and supralethal doses of total body gamma-radiation.

Pseudomonas Can Survive Tailocin Killing via Persistence-Like and Heterogenous Resistance Mechanisms.

Phage tail-like bacteriocins (tailocins) are bacterially produced protein toxins that mediate competitive interactions between cocolonizing bacteria. Both theoretical and experimental research has shown there are intransitive interactions between bacteriocin-producing, bacteriocin-sensitive, and bacteriocin-resistant populations, whereby producers outcompete sensitive cells, sensitive cells outcompete resistant cells, and resistant cells outcompete producers. These so-called rock-paper-scissors dynamics explain how all three populations occupy the same environment, without one driving the others extinct. Using Pseudomonas syringae as a model, we demonstrate that otherwise sensitive cells survive bacteriocin exposure through a physiological mechanism. This mechanism allows cells to survive bacteriocin killing without acquiring resistance. We show that a significant fraction of the target cells that survive a lethal dose of tailocin did not exhibit any detectable increase in survival during a subsequent exposure. Tailocin persister cells were more prevalent in stationary- rather than log-phase cultures. Of the fraction of cells that gained detectable resistance, there was a range from complete (insensitive) to incomplete (partially sensitive) resistance. By using genomic sequencing and genetic engineering, we showed that a mutation in a hypothetical gene containing 8 to 10 transmembrane domains causes tailocin high persistence and that genes of various glycosyltransferases cause incomplete and complete tailocin resistance. Importantly, of the several classes of mutations, only those causing complete tailocin resistance compromised host fitness. This result indicates that bacteria likely utilize persistence to survive bacteriocin-mediated killing without suffering the costs associated with resistance. This research provides important insight into how bacteria can escape the trap of fitness trade-offs associated with gaining de novo tailocin resistance.IMPORTANCE Bacteriocins are bacterially produced protein toxins that are proposed as antibiotic alternatives. However, a deeper understanding of the responses of target bacteria to bacteriocin exposure is lacking. Here, we show that target cells of Pseudomonas syringae survive lethal bacteriocin exposure through both physiological persistence and genetic resistance mechanisms. Cells that are not growing rapidly rely primarily on persistence, whereas those growing rapidly are more likely to survive via resistance. We identified various mutations in lipopolysaccharide biogenesis-related regions involved in tailocin persistence and resistance. By assessing host fitness of various classes of mutants, we showed that persistence and subtle resistance are mechanisms P. syringae uses to survive competition and preserve host fitness. These results have important implications for developing bacteriocins as alternative therapeutic agents.

A Lassa Virus Live-Attenuated Vaccine Candidate Based on Rearrangement of the Intergenic Region.

Lassa virus (LASV) poses a significant public health problem within the regions of Lassa fever endemicity in Western Africa. LASV infects several hundred thousand individuals yearly, and a considerable number of Lassa fever cases are associated with high morbidity and lethality. No approved LASV vaccine is available, and current therapy is limited to an off-label usage of ribavirin that is only partially effective and associated with significant side effects. The impact of Lassa fever on human health, together with the limited existing countermeasures, highlights the importance of developing effective vaccines against LASV. Here, we present the development and characterization of a recombinant LASV (rLASV) vaccine candidate [rLASV(IGR/S-S)], which is based on the presence of the noncoding intergenic region (IGR) of the small (S) genome segment (S-IGR) in both large (L) and S LASV segments. In cultured cells, rLASV(IGR/S-S) was modestly less fit than wild-type rLASV (rLASV-WT). rLASV(IGR/S-S) was highly attenuated in guinea pigs, and a single subcutaneous low dose of the virus completely protected against otherwise lethal infection with LASV-WT. Moreover, rLASV(IGR/S-S) was genetically stable during serial passages in cultured cells. These findings indicate that rLASV(IGR/S-S) can be developed into a LASV live-attenuated vaccine (LAV) that has the same antigenic composition as LASV-WT and a well-defined mechanism of attenuation that overcomes concerns about increased virulence that could be caused by genetic changes in the LAV during multiple rounds of multiplication.IMPORTANCE Lassa virus (LASV), the causative agent of Lassa fever, infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever cases. No U.S. Food and Drug Administration-licensed countermeasures are available to prevent or treat LASV infection. We describe the generation of a novel LASV live-attenuated vaccine candidate rLASV(IGR/S-S), which is based on the replacement of the large genomic segment noncoding intergenic region (IGR) with that of the small genome segment. rLASV(IGR/S-S) is less fit in cell culture than wild-type virus and does not cause clinical signs in inoculated guinea pigs. Importantly, rLASV(IGR/S-S) protects immunized guinea pigs against an otherwise lethal exposure to LASV.

Mortality of Different Stages of Plodia interpunctella (Lepidoptera: Pyralidae) at Three Temperatures in Controlled Atmosphere of High Nitrogen.

The lethal exposure time to controlled atmospheres of high nitrogen at stored grain temperatures is an important information for control of stored-product insects. The mortality of 1-d-old egg, 1-wk-old (first or second instar) larva, 3-wk-old (fourth or fifth instar) larva, and 1-d-old pupa of Plodia interpunctella (Hübner) was determined at 18 ± 1, 23 ± 1, and 28 ± 1°C in 98% N2 mixed with air. At 18°C, the lethal exposure times to achieve 100% mortality were 12.7 ± 0.7, 16.3 ± 0.3, 19.7 ± 0.7, and 14.7 ± 0.7 d for 1-d-old egg, 1-wk-old larva, 3-wk-old larva, and 1-d-old pupa, respectively. Temperature had significant effect on the lethal exposure time, and increase of the temperature significantly decreased the lethal exposure time. The order of the insect stages from the highest to lowest for LT50 values was follows: 3-wk-old larva > 1-wk-old larva > 1-d-old pupa ≥ 1-d-old egg. The minimum lethal exposure times required to kill all stages of P. interpunctella were about 20, 16, and 12 d at 18, 23, and 28°C, respectively.

A Lassa Fever Live-Attenuated Vaccine Based on Codon Deoptimization of the Viral Glycoprotein Gene.

Lassa virus (LASV) is endemic in Western Africa and is estimated to infect hundreds of thousands of individuals annually. A considerable number of these infections result in Lassa fever (LF), which is associated with significant morbidity and a case-fatality rate as high as 69% among hospitalized confirmed patients. U.S. Food and Drug Administration-approved LF vaccines are not available. Current antiviral treatment is limited to off-label use of a nucleoside analogue, ribavirin, that is only partially effective and associated with significant side effects. We generated and characterized a recombinant LASV expressing a codon-deoptimized (CD) glycoprotein precursor gene (GPC), rLASV-GPC/CD. Comparison of growth kinetics and peak titers showed that rLASV-GPC/CD is slightly attenuated in cell culture compared to wild-type (WT) recombinant LASV (rLASV-WT). However, rLASV-GPC/CD is highly attenuated in strain 13 and Hartley guinea pigs, as reflected by the absence of detectable clinical signs in animals inoculated with rLASV-GPC/CD. Importantly, a single subcutaneous dose of rLASV-GPC/CD provides complete protection against an otherwise lethal exposure to LASV. Our results demonstrate the feasibility of implementing a CD approach for developing a safe and effective LASV live-attenuated vaccine candidate. Moreover, rLASV-GPC/CD might provide investigators with a tool to safely study LASV outside maximum (biosafety level 4) containment, which could accelerate the elucidation of basic aspects of the molecular and cell biology of LASV and the development of novel LASV medical countermeasures.IMPORTANCE Lassa virus (LASV) infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever (LF) cases. Licensed LF vaccines are not available, and anti-LF therapy is limited to off-label use of the nucleoside analog ribavirin with uncertain efficacy. We describe the generation of a novel live-attenuated LASV vaccine candidate. This vaccine candidate is based on mutating wild-type (WT) LASV in a key region of the viral genome, the glycoprotein precursor (GPC) gene. These mutations do not change the encoded GPC but interfere with its production in host cells. This mutated LASV (rLASV-GPC/CD) behaves like WT LASV (rLASV-WT) in cell culture, but in contrast to rLASV-WT, does not cause disease in inoculated guinea pigs. Guinea pigs immunized with rLASV-GPC/CD were protected against an otherwise lethal exposure to WT LASV. Our results support the testing of this candidate vaccine in nonhuman primate models ofLF.

Variation in growth and evaluation of cross‐protection in Listeria monocytogenes under salt and bile stress

Exposure of Listeria monocytogenes to osmotic stress can induce increased resistance to subsequent lethal exposure to cell envelope stressors, such as nisin and bile salts. We wanted to determine if similar cross-protection phenotypes could occur when L. monocytogenes strains were treated with osmotic stress and exposed to sublethal levels of the cell envelope stressor, bile. Growth phenotypes were measured for six L. monocytogenes strains exposed to 6% NaCl, 0·3 and 1% bile in BHI. To evaluate cross-protection, cells were pre-exposed to 6% NaCl, followed by exposure to BHI+1% bile for 26h and vice versa. Significant increases in λ (lag phase) and doubling time were observed under salt and bile stresses compared with BHI alone. Average λ and Nmax (maximum cell density) in 0·3 and 1% bile for all strains were significantly lower than that in 6% NaCl. Pre-exposure to 6% NaCl followed by exposure to 1% bile significantly increased λ (P<0·05), whereas pre-exposure to 1% bile followed by exposure to 6% NaCl led to formation of filamentous cells, with no changes in cell density over 26h. Variation in growth characteristics was observed among strains exposed to bile. Exposure to osmotic stress did not lead to increased resistance to bile. Exposure to bile significantly impacted the ability of L. monocytogenes to adapt to grow under osmotic stress, where cells did not multiply but formed filamentous cells. Pre-exposure to a cell envelope stress and subsequent exposure to an osmotic stress appears to pose a significant stress to L. monocytogenes cells.

Understanding the Effects of Sublethal Pesticide Exposure on Honey Bees: A Role for Probiotics as Mediators of Environmental Stress

Managed populations of the European honey bee (Apis mellifera) support the production of a global food supply. This important role in modern agriculture has rendered honey bees vulnerable to the noxious effects of anthropogenic stressors such as pesticides. Although the deleterious outcomes of lethal pesticide exposure on honey bee health and performance are apparent, the ominous role of sublethal pesticide exposure is an emerging concern as well. Here, we use a data harvesting approach to better understand the toxicological effects of pesticide exposure across the honey bee life cycle. Through compiling adult- and larval-specific median lethal dose (LD50) values from 93 published data sources, LD50 estimates for insecticides, herbicides, acaricides, and fungicides are highly variable across studies, especially for herbicides and fungicides, which are underrepresented in the meta-data set. Alongside major discrepancies in these reported values, further examination of the compiled data suggested that LD50 may not be an ideal metric for honey bee risk assessment. We also discuss how sublethal effects of pesticide exposure, which are not typically measured in LD50 studies, can diminish honey bee reproduction, immunity, cognition, and overall physiological functioning, leading to suboptimal honey bee performance and population reduction. In consideration of actionable solutions to mitigate the effects of sublethal pesticide exposure, we have identified the potential for probiotic supplementation as a promising strategy that can be easily incorporated alongside current agricultural infrastructure and apicultural management practices. Probiotic supplementation is regularly employed in apiculture but the potential for evidence-based targeted approaches has not yet been fully explored within a formal toxicological context. We discuss the benefits, practical considerations, and limitations for the use and delivery of probiotics to hives. Ultimately, by subverting the sublethal effects of pesticides we can help improve the long-term survival of these critical pollinators.